The central goal of this proposal is to elucidate the mechanism(s) by which B cell depletion therapy (BCDT) and other targeted B cell therapies are efficacious and restore tolerance in systemic lupus erythematosus (SLE). It is hypothesized that loss of tolerance in SLE is due to an alteration in the balance of B cell survival signals (e.g. BAFF-B cell activator of the TNF family) relative to the numbers of transitional B cells emerging from the bone marrow (BM) (high BAFF/low numbers of emerging BM B cells), decreasing the stringency of negative selection. It is further postulated that a key dysregulated signal is interferon (IFN), with local production in the BM inhibiting BM B cell lymphopoiesis. On the other hand, we have found that a subset of SLE patients treated with BCDT experience restoration of B cell tolerance and have a unique pattern of B cell reconstitution characterized by exuberant B cell lymphopoiesis and a prominent expansion of circulating transitional cells. Thus, restoration of homeostatic mechanisms within the B cell transitional compartment may be critical for tolerance restoration in this group of subjects and, moreover, transitional B cells may play a physiological regulatory role that is dysregulated in SLE and restored after BCDT. This model will be tested through the following specific aims: 1. Define the factors regulating homeostasis of the transitional B cell compartment in SLE with a focus on BM B cell lymphopoiesis; 2. Determine the consequences of B cell depletion therapy and targeted biologic antagonism of BAFF or IFN on transitional B cell homeostasis and tolerance; and 3. Elucidate the immunoregulatory roles of transitional B cells and the contribution of a transitional B cell expansion to disease improvement in SLE after BCDT. Specifically, BM lymphopoiesis and its regulation will be examined by multi-parameter flow cytometry of BM B cell subsets and delineation of the cytokine milieu (IFN BAFF via luminex, gene signature, and flow based signaling) in normal controls, untreated SLE, and SLE after BCDT, anti-BAFF, or anti-IFN therapy. The phenotype, survival, proliferation, and selection of transitional B cells emerging from the BM will be ascertained based on flow cytometry, annexin-V binding, proliferation antigen expression, and replication history relative to IFN and engagement by BAFF. Tolerance will be assessed as a decrease in the frequency of autoreactive B cells during maturation from the transitional to the mature stage using single cell PCR and ELISPOT technology. The ability of transitional B cells to express anti-inflammatory cytokines (IL10) and induce a T regulatory cell phenotype will be defined. These studies will illuminate the mechanisms that underlie SLE, the role of B cells in autoimmunity, and the ways in which targeted therapy may improve disease.